EFEK PROTEKTIF KOPI TERHADAP ATEROSKLEROSIS KORONER PADA MODEL TIKUS PERIODONTITIS
Bidang Unggulan: Ketahanan pangan secara luas
Kode/Nama Rumpun Ilmu: 304/Ilmu Biomedik
EXECUTIVE SUMMARY
PENELITIAN UNGGULAN PERGURUAN TINGGI
(TAHUN KEDUA)
KAJIAN EFEK KOPI DAN PERIODONTITIS PADA ATEROSKLEROSIS KORONER
SUB JUDUL:
PROTECTIVE EFFECT OF COFFEE AGAINST CORONARY ATHEROSCLEROSIS IN
PERIODONTITIS RAT MODEL
Tim Pengusul:
Dr. drg. I Dewa Ayu Susilawati, M. Kes. (0003096112)
dr. Suryono, Sp. JP, FIHA. (0011106913)
drg. Tantin Ermawati, M. Kes. (0022038007)
UNIVERSITAS JEMBER
NOVEMBER 2014
PROTECTIVE EFFECT OF COFFEE AGAINST CORONARY ATHEROSCLEROSIS
IN PERIODONTITIS RAT MODEL
1
I Dewa Ayu Susilawati; 2Suryono; 3Tantin Ermawati
1, 3
2
Biomedic Department Dentistry Faculty University of Jember, Indonesia
Department of Cardiology Faculty of Medicine University of Jember, Indonesia
E-mail: dewayususilawati@yahoo.com
Abstract
Objectives. Effect of habitual coffee consumption on cardiovascular disease is controversial.
Some epidemiological studies, however, reported the beneficial effect of coffee consumption on
cardiovascular health. The objective of this study was to conduct an in vivo experiment to prove
the effect of coffee consumption to reduce coronary atherosclerosis in periodontitis rat model.
Methods. Twenty one rats (Rattus norvegicus) were divided into three groups, i. e. 1)
periodontitis, 2) periodontitis + coffee, 3) control group. Periodontitis rat model was created by
means of inserting wire ligature around left molar mandibular teeth followed by injecting
periodontitis bacteria Porphyromonas gingivalis in gingival sulcus thrice a week. One dose of
decocta coffee (representing one cup) was fed once per day by stomach sondation. The
experiment was conducted for 35 days. At the end of experiment, all rats were sacrified. Their
hearts which contained coronary arteries were removed and prepared for histopathologic
examination for media-intimal thickening and ratio, the existence of atheroma, stenosis,
endothelial cell disintegration and lipids deposit. Results. Arterial wall of rats that consumed
coffee demonstrated more symmetric media-intimal thickness and intimal collagen showed
intact, denser and thicker. Fewer stenosis, atheroma, lipid deposit and endothelial cell
disintegration were identified in coffee group. Conclusions. One cup coffee consumption per
day improved the morphology of coronary artery leading to protect against atherosclerosis. A
novel perspective was the improvement of the structure of intimal collagen, it might also provide
the resistance of vasculature against rupture and thrombosis, further studies are needed
particularly focusing on this intimal collagen.
Keywords. Atherosclerosis; Coffee; Collagen; Periodontitis; Rat.
EFEK PROTEKTIF KOPI TERHADAP ATEROSKLEROSIS KORONER
PADA MODEL TIKUS PERIODONTITIS
Abstrak
Latar belakang. Pengaruh kebiasaan minum kopi pada penyakit kardiovaskular masih kontroversial.
Beberapa studi epidemiologi, melaporkan efek positif dari konsumsi kopi pada kesehatan jantung.
Tujuan dari penelitian ini adalah untuk melakukan percobaan in vivo untuk membuktikan pengaruh
konsumsi kopi untuk mengurangi aterosklerosis koroner pada model tikus periodontitis. Metode. Dua
puluh satu tikus (Rattus norvegicus) dibagi menjadi tiga kelompok, i. e. 1) periodontitis, 2) periodontitis +
kopi, 3) kelompok kontrol. Model tikus periodontitis dibuat dengan cara mengikatkan kawat ligatur di
sekitar gigi molar rahang bawah, diikuti dengan menyuntikkan bakteri periodontitis Porphyromonas
gingivalis pada sulkus gingiva tiga kali seminggu. Satu dosis seduan kopi (mewakili satu cangkir)
diberikan sekali per hari melalui sondasi lambung. Penelitian dilakukan selama 35 hari. Pada akhir
percobaan, semua tikus dikorbankan. Jantung mereka yang berisi arteri koroner diambil dan disiapkan
untuk pemeriksaan histopatologi untuk dianalisa penebalan media intima dan rasio, keberadaan ateroma,
stenosis, disintegrasi sel endotel dan deposito lemak. Hasil. Arteri dinding tikus yang mengkonsumsi kopi
menunjukkan morfologi ketebalan media intima lebih simetris dan kolagen intima tampak utuh, lebih
padat dan lebih tebal. Sedikit stenosis, ateroma, deposisi lemak dan disintegrasi sel endotel diidentifikasi
dalam kelompok kopi. Kesimpulan. Konsumsi secangkir kopi per hari memperbaiki morfologi arteri
koroner sehingga melindungi terhadap aterosklerosis. Perspektif baru pada penelitian ini adalah
perbaikan struktur kolagen intima, hal ini mungkin juga menjelaskan ketahanan pembuluh darah terhadap
ruptur dan trombosis, studi lebih lanjut diperlukan terutama berfokus pada intima kolagen ini.
Kata kunci: Aterosklerosis; Kopi; Kolagen; Periodontitis; Tikus.
INTRODUCTION
Perception on effect of coffee beverage consumption on cardiovascular disease has changed
[Lopez Garcia, 2012]. Several earlier studies had shown the adverse effect of coffee
consumption on cardiovascular system, such as, related to the risk of acute coronary events
[LaCroix 1986, Happonen 2004], cause extracellular accumulation of low density lipoprotein
(LDL) [Rustam, 1997], increased inflammation [Zampelas, 2004], heightened acute vascular
inflammatory (Hammer, 2006), induced unfavorable effects on
endothelial function
[PAPAMICHAE , Buscemi]. Results from more recent studies, however, reported beneficial of
habitual coffee consumption on cardiovascular health, such as effects on subclinical
inflammation and HDL cholesterol [Kempt, 2010], against coronary calcification [Geertruida J.
van Woudenbergh 2008], inversely associated with markers of inflammation and endothelial
dysfunction [Lopez-Garcia E 2006], reduce risk of death attributed to inflammatory and
cardiovascular disease [Anderson, 2006], inversely associated with risk of heart failure
[Mostofski, 2012] improved endothelial function [Shecher, 2011; Siosa 2013], increase the
resistance of LDL to oxidative modification [Natella, 2007].
Many studies on coffee concerned with the harmful effect of caffeine used in isolation [Adebayo,
2006; Corneli 2007; Silvas 2004], in fact, coffee has a very complex chemical composition. In
addition to caffeine, coffee contained many other substances that act as antioxidants [Natella
2007; Silvas 2004; Halvas 2002; Moriera 2012] and anti-inflammatory [Kempt 2010; Anderson
2006; moriera 2012], that may counteract the adverse effect of coffee. Considering these
properties of antioxidant and anti-inflammatory, therefore it is plausible to assume that coffee
consumption may have protective effect against atherosclerosis. Since, oxidation and
inflammation represent important mechanism in atherogenesis [Fong 2002].
The currently accepted hypothesis is that atherosclerosis develops as a response to injury and that
is primarily a chronic inflammatory condition [stocker]. Chronic bacterial inflammatory disease
of tooth surrounding tissue (periodontitis) had been shown to play an important role in
pathogenesis of atherosclerosis. Periodontitis bacteries, were identified using immunostaining
and polymerase chain reaction (PCR) in some autopsy specimen of coronary atherosclerotic
plaque in human who died for heart attack [Harazthy, Kozarov, stelzel]. Animal experimental
studies consistently shown the positive role of periodontitis in increase risk of atherosclerosis [li,
brodala].
The purpose of this study was to conduct an in vivo experiment to prove the effect of coffee
consumption to reduce coronary atherosclerosis, in periodontitis rat model. In particular, the
present study analyzed the effect of one dose of coffee decocta consumption (representing one
cup per day). Indicators of coronary atherosclerosis included diffuse intimal thickening (DIT),
morphological change of arterial wall, lipids deposit, foam cell, and scavenger receptor (Sc-R)
expression.
METHODS
Animals and groups
Male rats (Rattus norvegicus), 12 weeks old (± 200 gr body weight) were purchased from the
Department of Physiology Faculty of Medicine Brawijaya University, Malang, Indonesia. The
animal were maintained under standard laboratory conditions and according to the guidelines
established by institutional Animal Care and Ethics Committee. Animal treatment procedure was
approved by Ethical committee at Faculty of Dentistry Gadjah Mada University, Yogyakarta,
Indonesia. Dry pellet normocholesterol standard diet and water were given ad libitum. All rats
were housed in pens in identical condition to minimize environment factor. Total of twenty one
rats were randomly divided into three groups, namely 1) periodontitis, 2) periodontitis + coffee
(coffee group), and 3) control (healthy, non-periodontitis, no coffee).
Preparation of coffee decocta
This study used commercial pure Robusta ground coffee produced by State Plantation Company
PTPN XII Jember, East Java, Indonesia. To prepare coffee decocta, 3 gr ground coffee was
poured into 200 ml boiled aquades, for 3 minutes, while shaking. One dose of coffee decocta
representing one cup (0,6 ml) was given per day to rats by means of stomach sondation.
Periodontitis rat model.
Periodontitis model was created in rat by means injecting periodontitis bacteria Porphyromonas
gingivalis (ATTC, 33277) in buccal gingival sulcus of left mandibular teeth, as previously
described (Susilawati). Before injection, these teeth were tided using a loop wire ligature (θ: 0,5
mm, Fried Krupp) in order to make better retention for bacterial plaque to accumulate on
periodontal tissue leading to accelerate periodontitis. This procedure was performed under
intramuscular anesthesia using ketamin 0,02ml/rat. P. gingivalis was prepared in cultured
medium Brain Heart Infusion Broth (BHI-B) enriched by vitamin K1 and hemin (Sigma) under
anaerobic atmosphere for 2 x 24 hours. To confirm bacterial purity, P. gingivalis was stained
using Gram dye, and identification under light microscope shown that P. gingivalis was Gram
negative with uniform rod morphology. P. gingivalis concentration was adjusted to Mack
Farland 0,5 (1,5 x 108) CFU per ml. Chronic periodontitis was conditioned by regular injection
of 50 μl P. gingivalis thrice a week for five weeks (35 days). At the end of study, all of the rats
in periodontitis group demontrated alveolar bone resorption clinically and radiographically
(suffered from periodontitis).
Histologic samples preparation
At the end of experiment, all of rats were fasted overnight and afterword were sacrified using a
heavily chloroform inhalation. The heart which contained coronary arteries were removed and
fixed in 10% formalin (in PBS), and then trimmed crossectionally (perpendicular to the direction
of blood flow) in about coronal one half area of the heart, and prepared for frozen section. Serial
cryosections (10 μm thickness) were made and then mounted on to microscope slides
Coronary arterial wall thickness and morphology
To analyze coronary arterial wall thickness and morphology, samples were stained using
collagen staining kit (Picro’Siruis Red). According to the instruction of the manufacturer, this
stain demonstrated red color for collagen and yellow for elastin. The images were demonstrated
by light microscope magnification 400 and 1000 times and visualized using optilab viewer.
Coronary arterial wall thickness was measured as media-intima thickness (μm), in the thickest
area. We also observed focusing on intimal collagen integrity and thickness. In addition,
symmetric and asymmetric thickness and media-intima ratio were analyzed as well. For
morphologic parameters, we identified the existence of irregularity, atheroma, stenosis, luminal
narrowing and occlusion.
Endothelial cell layer integration and lipid deposit
Morphology of endothelial layer were analyzed in histologic samples after being stained by
Hematoxylin-Eosin dye. Lipid deposit, foam cell and fatty emboli were identified in frozen
section slide that were stained by Oil RedO dye (Sigma) and hematoxyline (Merck) for counter
stain. 5% Oil RedO was prepared by dilution in propilene glikol. Lipid deposit was demonstrated
as red color droplet, while nuclei pale blue.
Analysis of intimal collagen integrity
Integrity of intimal collagen is important to maintain the resistance of vasculature and
atherosclerotic plaque against rupture and thrombosis. Disintegration of intimal collagen could
cause thinning of fibrous cap so that increase the risk of rupture. The present study analyzed
quality intimal collagen such as the intact, dense and disintegration. This was analyzed in
samples that were stained by collagen staining kit.
RESULTS
Coronary Arterial Wall Thickness
Analysis using collagen staining method clearly demonstrated the comparation of coronary
arterial thickness between groups. Coffee group showed thinner arterial wall compared to non
coffee groups. This was confirmed by figure 2, that demonstrated an obvious arterial wall
thickening in periodontitis group (B) compared to coffee (C) and non-periodontitis group (A). In
addition, We also found that coffee group have more symmetric intimal thickness and relatif
wider lumen and minimum blood component adhesion in the luminal surface.
Coronary Arterial Wall Morphology
Further analysis of coronary arterial wall morphology using collagen staining method was
focused on the morphology of intimal layer. A significant difference on intimal layer
morphology depicted on Figure 3. Collagen of endothelial basal membrane in intimal layer of
coffee group showed obviously thickest and intact (Figure 3C) compared to other groups. In
periodontitis group, collagen basal membrane were thinnest and disintegrated (Figure 3B), while
in non periodontitis group were shown as a thin layer (Figure 3A).
Expression of Scavenger Receptor
Immunohistochemical analysis of coronary artery revealed that the expression of scavenger
receptor (CD 163) were fewer in coffee and control groups compared to periodontitis group.
Figure 4, coronary artery of periodontitis rat model expressed more scavenger receptors.
Lipid Deposition
Histochemistry analysis of coronary artery using Oil RedO demonstrated that endothelial lipid
deposition, fatty emboli and foam cell were more common found in periodontitis group
compared to control and coffee groups. Rat that fed with coffee showed minimum endothelial
lipid deposition and fatty emboli was absent.
Figure 5. Lipid deposition (demonstrated by Oil RedO staning) on endothelial surface and fatty
emboli were found in coronary artery of periodontitis rat model (B) compared to control group
(A). Fewest lipid deposition and fatty emboli were identified in coffee group (C).
DISCUSSION
The present study revealed that chronic periodontitis induced worsening morphology of coronary
arterial wall leading to accelerate atherosclerosis. Several atherosclerosis parameters identified
were arterial wall thickening, progression atheroma morphology, endothelial lipid deposition and
disintegration of intimal collagen. This result is consistent with our previous study that
atherosclerotic lesion were identified more frequently in periodontitis rat model (Susilawati, fdi
yogya). Other authors reported that experimental bacteremia (Bordala, Lie) using periodontitis
bacteria P. gingivalis accelerated atherosclerosis. In the present study, instead of direct
bacteremia, systemic inflammation was assumed to be originated from chronic periodontal focal
infection (chronic infection was maintained by regular injecting P. gingivalis di periodontal
tissue). The level of bacteremia and systemic inflammation, however, in this study was not
measured.
The deleterious effect of chronic periodontitis in coronary artery could be encountered by means
of habitual one cup coffee consumption per day, in which protected against coronary
atherosclerosis. Rats that consumed coffee demonstrated more symmetric arterial wall thickness,
no atheroma progression, fewer endothelial lipid deposition and fatty emboli. Most importance
result was the effect on the morphology of intimal collagen, in coffee group, coronary intimal
collagen was intact and significantly denser and thicker compared to control and periodontitis
groups. This might explain the resistance of the vasculature against injurious agents from blood
stream, so that reduced the response to injury leading to reduce atherosclerosis. This is a new
perspective. This is the first report of the beneficial effect of coffee on improving vasculature
intimal collagen.
The integrity of vasculature intimal collagen is modulated by the activation of matrix degrading
enzymes, matrix metalloproteinases (MMPs). These enzymes are mainly produced by
inflammatory cells as inactive zymogen (proMMPs). Some activators such as bacterial
proteinases and oxidants/free radicals can be activated proMMPs to become active MMPs by
mechanism of proteolysis and cysteine switch, respectively. Considering condition of systemic
infection, We speculated that entering of injurious agents into blood circulation could stimulate
inflammatory response in vasculature such as production of large amount of proMMPs, oxidants
and free radicals, and interaction of these substances reveal active MMPs that degrade
vasculature collagen.
Pathologic degradation of vascular collagen is the basis molecular process for atherosclerotic
plaque rupture leading to coronary thrombosis and acute coronary syndrome (Shah 2005; Gough,
2006) It was constantinides (1994) who firstly noticed that arterial thrombosis is practically
always initiated by local breaks of vascular collagen. When there is vascular collagen
breakdown, platelet will encounter and readily to aggregate. Platelet aggregation will be
followed by activation of coagulation cascade leading to subsequent thrombus formation.3 When
thrombosis is occlusive and happened on atherosclerotic plaque near by coronary artery it could
stop up the blood supply into the heart leading to ischemia and heart attack.4 Hence, pathologic
vascular collagen degradation is a critical event determining the clinical symptom of ACS.
There was speculated that the benefit effect of coffee was due to antioxidant and antiinflammatory compounds, such as polyphenol, that is rich in coffee [Moreira]. The effect of
polyphenol-rich food or polyphenol extracts on development of atherosclerotic lesion had been
studied in animal model [mura, Vinson, dufi, manach, bernatova]. Polyphenol improved function
of endothelial cell and inhibit platelet aggregation (Vita 2005), decreased oxidation of LDL [de
whalley, Natella], improved cardiovascular remodeling and vascular function [bertanova],
increased plasma antioxidant carotenoid [svilaas]. In the present study, we speculated that
antioxidant and anti-inflammatory compounds in coffee might modulate inflammatory response
and enzymes that responsible for collagen degradation, it is matrix metalloproteinases (MMPs).
Matrix metalloproteinases is produced as inactive enzymes (zymogen) proMMPs, their
activation depends on any activators such as proteinases and or reactive oxygen species (ROS).
Inflammatory response increased production of inflammatory cytokines such as TNF-α, IL-1β
that can induce expression of proMMPs. In addition, bacterial infection induced large amount of
ROS that could activate proMMP by mechanism of cysteine switch. Therefore interaction
components in inflammation could result in MMP activation leading to degrade collagen.
The most significant result of this study was the influence of coffee consumption on the
morphology of collagen intimal. It may open a novel perspective study. There is only a few study
or literatures that mention the role of coffee in collagen metabolism, particularly vascular
collagen. Further studies are needed to elucidate this mechanism effect of coffee in collagen
metabolism, in addition, the influence of its structure to cardiovascular function as well. Like as
mention above, in addition to caffeine, coffee is a mixture of thousand substances including
antioxidants. There is still unclear, whether the effect of coffee on collagen metabolism was
mediated by its antioxidant, many works however are still needed to elucidate the role of
antioxidants in collagen metabolism.
The clinical significant of coronary artery morphology that composed by intact and dense intimal
collagen might provide protective effect against atherosclerosis and rupture. Since
atherosclerotic plaque rupture mostly occurred in area of thin collagen fibrous cap [Gouge,
2006]. Atherosclerotic plaque rupture is the proximate event of clinical manifestation of acute
coronary syndrome. Therefore it can be stated that coffee might prevent the incidence of acute
coronary syndrome by improving the morphology of arterial wall particularly intimal collagen.
Cardiovascular effect of coffee is necessary to continue to be explored. Although some studies
revealed the positive cardiovascular effect, many studies however suggested the adverse effect.
There is still common perception that caffeine in coffee can cause blood pressure to increase. This
is due to the effect of coffee to increase adrenal glands to release more adrenaline, unfavorable effect on
endothelial function (Papamitchel 2006, buschemy 2010) In addition to chemical compositions, variety
factors could influence the effect of coffee, such as frequency of consumption, type of coffee
procecesing, brewing method, host factors (e. g. age, gender), etc.
The present study use pure one dose of coffee decocta per day that represent one cup of coffee.
Further study are needed to analyze effect of the dosis, since some study indicated that high
intake of coffee have negative effect on cardiovascular health. In addition to dosis, coffee
serving usually was mixed with other ingredients such as milk and sugar. There still not known
whether coffee-nutrient interaction would influence the effect of coffee. Other factors like high
lipid diet, alcohol consumption, smoking are also commonly follow the habitual coffee
consumption are needed to study as well.
In conclusion, coffee consumption improved morphology of arterial wall leading to protection
against coronary atherosclerosis. This protective effect might also explain the resistance
mechanism of vasculature against rupture, thrombosis, and it could be meant that coffee
consumption could reduce the incidence of acute coronary syndrome. Most interestingly, this
study provide evidence that coffee consumption influence the structure of intimal collagen of
coronary artery. Further studies will be focused on elucidation of factors that affect its collagen
structure and composition.
Acknowledgement
This study was funding by University developmental grant of Jember University. Special thank
to Mbak Wahyu, Mas Agus and Mas Pinardi for helping us in laboratory works.
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Kode/Nama Rumpun Ilmu: 304/Ilmu Biomedik
EXECUTIVE SUMMARY
PENELITIAN UNGGULAN PERGURUAN TINGGI
(TAHUN KEDUA)
KAJIAN EFEK KOPI DAN PERIODONTITIS PADA ATEROSKLEROSIS KORONER
SUB JUDUL:
PROTECTIVE EFFECT OF COFFEE AGAINST CORONARY ATHEROSCLEROSIS IN
PERIODONTITIS RAT MODEL
Tim Pengusul:
Dr. drg. I Dewa Ayu Susilawati, M. Kes. (0003096112)
dr. Suryono, Sp. JP, FIHA. (0011106913)
drg. Tantin Ermawati, M. Kes. (0022038007)
UNIVERSITAS JEMBER
NOVEMBER 2014
PROTECTIVE EFFECT OF COFFEE AGAINST CORONARY ATHEROSCLEROSIS
IN PERIODONTITIS RAT MODEL
1
I Dewa Ayu Susilawati; 2Suryono; 3Tantin Ermawati
1, 3
2
Biomedic Department Dentistry Faculty University of Jember, Indonesia
Department of Cardiology Faculty of Medicine University of Jember, Indonesia
E-mail: dewayususilawati@yahoo.com
Abstract
Objectives. Effect of habitual coffee consumption on cardiovascular disease is controversial.
Some epidemiological studies, however, reported the beneficial effect of coffee consumption on
cardiovascular health. The objective of this study was to conduct an in vivo experiment to prove
the effect of coffee consumption to reduce coronary atherosclerosis in periodontitis rat model.
Methods. Twenty one rats (Rattus norvegicus) were divided into three groups, i. e. 1)
periodontitis, 2) periodontitis + coffee, 3) control group. Periodontitis rat model was created by
means of inserting wire ligature around left molar mandibular teeth followed by injecting
periodontitis bacteria Porphyromonas gingivalis in gingival sulcus thrice a week. One dose of
decocta coffee (representing one cup) was fed once per day by stomach sondation. The
experiment was conducted for 35 days. At the end of experiment, all rats were sacrified. Their
hearts which contained coronary arteries were removed and prepared for histopathologic
examination for media-intimal thickening and ratio, the existence of atheroma, stenosis,
endothelial cell disintegration and lipids deposit. Results. Arterial wall of rats that consumed
coffee demonstrated more symmetric media-intimal thickness and intimal collagen showed
intact, denser and thicker. Fewer stenosis, atheroma, lipid deposit and endothelial cell
disintegration were identified in coffee group. Conclusions. One cup coffee consumption per
day improved the morphology of coronary artery leading to protect against atherosclerosis. A
novel perspective was the improvement of the structure of intimal collagen, it might also provide
the resistance of vasculature against rupture and thrombosis, further studies are needed
particularly focusing on this intimal collagen.
Keywords. Atherosclerosis; Coffee; Collagen; Periodontitis; Rat.
EFEK PROTEKTIF KOPI TERHADAP ATEROSKLEROSIS KORONER
PADA MODEL TIKUS PERIODONTITIS
Abstrak
Latar belakang. Pengaruh kebiasaan minum kopi pada penyakit kardiovaskular masih kontroversial.
Beberapa studi epidemiologi, melaporkan efek positif dari konsumsi kopi pada kesehatan jantung.
Tujuan dari penelitian ini adalah untuk melakukan percobaan in vivo untuk membuktikan pengaruh
konsumsi kopi untuk mengurangi aterosklerosis koroner pada model tikus periodontitis. Metode. Dua
puluh satu tikus (Rattus norvegicus) dibagi menjadi tiga kelompok, i. e. 1) periodontitis, 2) periodontitis +
kopi, 3) kelompok kontrol. Model tikus periodontitis dibuat dengan cara mengikatkan kawat ligatur di
sekitar gigi molar rahang bawah, diikuti dengan menyuntikkan bakteri periodontitis Porphyromonas
gingivalis pada sulkus gingiva tiga kali seminggu. Satu dosis seduan kopi (mewakili satu cangkir)
diberikan sekali per hari melalui sondasi lambung. Penelitian dilakukan selama 35 hari. Pada akhir
percobaan, semua tikus dikorbankan. Jantung mereka yang berisi arteri koroner diambil dan disiapkan
untuk pemeriksaan histopatologi untuk dianalisa penebalan media intima dan rasio, keberadaan ateroma,
stenosis, disintegrasi sel endotel dan deposito lemak. Hasil. Arteri dinding tikus yang mengkonsumsi kopi
menunjukkan morfologi ketebalan media intima lebih simetris dan kolagen intima tampak utuh, lebih
padat dan lebih tebal. Sedikit stenosis, ateroma, deposisi lemak dan disintegrasi sel endotel diidentifikasi
dalam kelompok kopi. Kesimpulan. Konsumsi secangkir kopi per hari memperbaiki morfologi arteri
koroner sehingga melindungi terhadap aterosklerosis. Perspektif baru pada penelitian ini adalah
perbaikan struktur kolagen intima, hal ini mungkin juga menjelaskan ketahanan pembuluh darah terhadap
ruptur dan trombosis, studi lebih lanjut diperlukan terutama berfokus pada intima kolagen ini.
Kata kunci: Aterosklerosis; Kopi; Kolagen; Periodontitis; Tikus.
INTRODUCTION
Perception on effect of coffee beverage consumption on cardiovascular disease has changed
[Lopez Garcia, 2012]. Several earlier studies had shown the adverse effect of coffee
consumption on cardiovascular system, such as, related to the risk of acute coronary events
[LaCroix 1986, Happonen 2004], cause extracellular accumulation of low density lipoprotein
(LDL) [Rustam, 1997], increased inflammation [Zampelas, 2004], heightened acute vascular
inflammatory (Hammer, 2006), induced unfavorable effects on
endothelial function
[PAPAMICHAE , Buscemi]. Results from more recent studies, however, reported beneficial of
habitual coffee consumption on cardiovascular health, such as effects on subclinical
inflammation and HDL cholesterol [Kempt, 2010], against coronary calcification [Geertruida J.
van Woudenbergh 2008], inversely associated with markers of inflammation and endothelial
dysfunction [Lopez-Garcia E 2006], reduce risk of death attributed to inflammatory and
cardiovascular disease [Anderson, 2006], inversely associated with risk of heart failure
[Mostofski, 2012] improved endothelial function [Shecher, 2011; Siosa 2013], increase the
resistance of LDL to oxidative modification [Natella, 2007].
Many studies on coffee concerned with the harmful effect of caffeine used in isolation [Adebayo,
2006; Corneli 2007; Silvas 2004], in fact, coffee has a very complex chemical composition. In
addition to caffeine, coffee contained many other substances that act as antioxidants [Natella
2007; Silvas 2004; Halvas 2002; Moriera 2012] and anti-inflammatory [Kempt 2010; Anderson
2006; moriera 2012], that may counteract the adverse effect of coffee. Considering these
properties of antioxidant and anti-inflammatory, therefore it is plausible to assume that coffee
consumption may have protective effect against atherosclerosis. Since, oxidation and
inflammation represent important mechanism in atherogenesis [Fong 2002].
The currently accepted hypothesis is that atherosclerosis develops as a response to injury and that
is primarily a chronic inflammatory condition [stocker]. Chronic bacterial inflammatory disease
of tooth surrounding tissue (periodontitis) had been shown to play an important role in
pathogenesis of atherosclerosis. Periodontitis bacteries, were identified using immunostaining
and polymerase chain reaction (PCR) in some autopsy specimen of coronary atherosclerotic
plaque in human who died for heart attack [Harazthy, Kozarov, stelzel]. Animal experimental
studies consistently shown the positive role of periodontitis in increase risk of atherosclerosis [li,
brodala].
The purpose of this study was to conduct an in vivo experiment to prove the effect of coffee
consumption to reduce coronary atherosclerosis, in periodontitis rat model. In particular, the
present study analyzed the effect of one dose of coffee decocta consumption (representing one
cup per day). Indicators of coronary atherosclerosis included diffuse intimal thickening (DIT),
morphological change of arterial wall, lipids deposit, foam cell, and scavenger receptor (Sc-R)
expression.
METHODS
Animals and groups
Male rats (Rattus norvegicus), 12 weeks old (± 200 gr body weight) were purchased from the
Department of Physiology Faculty of Medicine Brawijaya University, Malang, Indonesia. The
animal were maintained under standard laboratory conditions and according to the guidelines
established by institutional Animal Care and Ethics Committee. Animal treatment procedure was
approved by Ethical committee at Faculty of Dentistry Gadjah Mada University, Yogyakarta,
Indonesia. Dry pellet normocholesterol standard diet and water were given ad libitum. All rats
were housed in pens in identical condition to minimize environment factor. Total of twenty one
rats were randomly divided into three groups, namely 1) periodontitis, 2) periodontitis + coffee
(coffee group), and 3) control (healthy, non-periodontitis, no coffee).
Preparation of coffee decocta
This study used commercial pure Robusta ground coffee produced by State Plantation Company
PTPN XII Jember, East Java, Indonesia. To prepare coffee decocta, 3 gr ground coffee was
poured into 200 ml boiled aquades, for 3 minutes, while shaking. One dose of coffee decocta
representing one cup (0,6 ml) was given per day to rats by means of stomach sondation.
Periodontitis rat model.
Periodontitis model was created in rat by means injecting periodontitis bacteria Porphyromonas
gingivalis (ATTC, 33277) in buccal gingival sulcus of left mandibular teeth, as previously
described (Susilawati). Before injection, these teeth were tided using a loop wire ligature (θ: 0,5
mm, Fried Krupp) in order to make better retention for bacterial plaque to accumulate on
periodontal tissue leading to accelerate periodontitis. This procedure was performed under
intramuscular anesthesia using ketamin 0,02ml/rat. P. gingivalis was prepared in cultured
medium Brain Heart Infusion Broth (BHI-B) enriched by vitamin K1 and hemin (Sigma) under
anaerobic atmosphere for 2 x 24 hours. To confirm bacterial purity, P. gingivalis was stained
using Gram dye, and identification under light microscope shown that P. gingivalis was Gram
negative with uniform rod morphology. P. gingivalis concentration was adjusted to Mack
Farland 0,5 (1,5 x 108) CFU per ml. Chronic periodontitis was conditioned by regular injection
of 50 μl P. gingivalis thrice a week for five weeks (35 days). At the end of study, all of the rats
in periodontitis group demontrated alveolar bone resorption clinically and radiographically
(suffered from periodontitis).
Histologic samples preparation
At the end of experiment, all of rats were fasted overnight and afterword were sacrified using a
heavily chloroform inhalation. The heart which contained coronary arteries were removed and
fixed in 10% formalin (in PBS), and then trimmed crossectionally (perpendicular to the direction
of blood flow) in about coronal one half area of the heart, and prepared for frozen section. Serial
cryosections (10 μm thickness) were made and then mounted on to microscope slides
Coronary arterial wall thickness and morphology
To analyze coronary arterial wall thickness and morphology, samples were stained using
collagen staining kit (Picro’Siruis Red). According to the instruction of the manufacturer, this
stain demonstrated red color for collagen and yellow for elastin. The images were demonstrated
by light microscope magnification 400 and 1000 times and visualized using optilab viewer.
Coronary arterial wall thickness was measured as media-intima thickness (μm), in the thickest
area. We also observed focusing on intimal collagen integrity and thickness. In addition,
symmetric and asymmetric thickness and media-intima ratio were analyzed as well. For
morphologic parameters, we identified the existence of irregularity, atheroma, stenosis, luminal
narrowing and occlusion.
Endothelial cell layer integration and lipid deposit
Morphology of endothelial layer were analyzed in histologic samples after being stained by
Hematoxylin-Eosin dye. Lipid deposit, foam cell and fatty emboli were identified in frozen
section slide that were stained by Oil RedO dye (Sigma) and hematoxyline (Merck) for counter
stain. 5% Oil RedO was prepared by dilution in propilene glikol. Lipid deposit was demonstrated
as red color droplet, while nuclei pale blue.
Analysis of intimal collagen integrity
Integrity of intimal collagen is important to maintain the resistance of vasculature and
atherosclerotic plaque against rupture and thrombosis. Disintegration of intimal collagen could
cause thinning of fibrous cap so that increase the risk of rupture. The present study analyzed
quality intimal collagen such as the intact, dense and disintegration. This was analyzed in
samples that were stained by collagen staining kit.
RESULTS
Coronary Arterial Wall Thickness
Analysis using collagen staining method clearly demonstrated the comparation of coronary
arterial thickness between groups. Coffee group showed thinner arterial wall compared to non
coffee groups. This was confirmed by figure 2, that demonstrated an obvious arterial wall
thickening in periodontitis group (B) compared to coffee (C) and non-periodontitis group (A). In
addition, We also found that coffee group have more symmetric intimal thickness and relatif
wider lumen and minimum blood component adhesion in the luminal surface.
Coronary Arterial Wall Morphology
Further analysis of coronary arterial wall morphology using collagen staining method was
focused on the morphology of intimal layer. A significant difference on intimal layer
morphology depicted on Figure 3. Collagen of endothelial basal membrane in intimal layer of
coffee group showed obviously thickest and intact (Figure 3C) compared to other groups. In
periodontitis group, collagen basal membrane were thinnest and disintegrated (Figure 3B), while
in non periodontitis group were shown as a thin layer (Figure 3A).
Expression of Scavenger Receptor
Immunohistochemical analysis of coronary artery revealed that the expression of scavenger
receptor (CD 163) were fewer in coffee and control groups compared to periodontitis group.
Figure 4, coronary artery of periodontitis rat model expressed more scavenger receptors.
Lipid Deposition
Histochemistry analysis of coronary artery using Oil RedO demonstrated that endothelial lipid
deposition, fatty emboli and foam cell were more common found in periodontitis group
compared to control and coffee groups. Rat that fed with coffee showed minimum endothelial
lipid deposition and fatty emboli was absent.
Figure 5. Lipid deposition (demonstrated by Oil RedO staning) on endothelial surface and fatty
emboli were found in coronary artery of periodontitis rat model (B) compared to control group
(A). Fewest lipid deposition and fatty emboli were identified in coffee group (C).
DISCUSSION
The present study revealed that chronic periodontitis induced worsening morphology of coronary
arterial wall leading to accelerate atherosclerosis. Several atherosclerosis parameters identified
were arterial wall thickening, progression atheroma morphology, endothelial lipid deposition and
disintegration of intimal collagen. This result is consistent with our previous study that
atherosclerotic lesion were identified more frequently in periodontitis rat model (Susilawati, fdi
yogya). Other authors reported that experimental bacteremia (Bordala, Lie) using periodontitis
bacteria P. gingivalis accelerated atherosclerosis. In the present study, instead of direct
bacteremia, systemic inflammation was assumed to be originated from chronic periodontal focal
infection (chronic infection was maintained by regular injecting P. gingivalis di periodontal
tissue). The level of bacteremia and systemic inflammation, however, in this study was not
measured.
The deleterious effect of chronic periodontitis in coronary artery could be encountered by means
of habitual one cup coffee consumption per day, in which protected against coronary
atherosclerosis. Rats that consumed coffee demonstrated more symmetric arterial wall thickness,
no atheroma progression, fewer endothelial lipid deposition and fatty emboli. Most importance
result was the effect on the morphology of intimal collagen, in coffee group, coronary intimal
collagen was intact and significantly denser and thicker compared to control and periodontitis
groups. This might explain the resistance of the vasculature against injurious agents from blood
stream, so that reduced the response to injury leading to reduce atherosclerosis. This is a new
perspective. This is the first report of the beneficial effect of coffee on improving vasculature
intimal collagen.
The integrity of vasculature intimal collagen is modulated by the activation of matrix degrading
enzymes, matrix metalloproteinases (MMPs). These enzymes are mainly produced by
inflammatory cells as inactive zymogen (proMMPs). Some activators such as bacterial
proteinases and oxidants/free radicals can be activated proMMPs to become active MMPs by
mechanism of proteolysis and cysteine switch, respectively. Considering condition of systemic
infection, We speculated that entering of injurious agents into blood circulation could stimulate
inflammatory response in vasculature such as production of large amount of proMMPs, oxidants
and free radicals, and interaction of these substances reveal active MMPs that degrade
vasculature collagen.
Pathologic degradation of vascular collagen is the basis molecular process for atherosclerotic
plaque rupture leading to coronary thrombosis and acute coronary syndrome (Shah 2005; Gough,
2006) It was constantinides (1994) who firstly noticed that arterial thrombosis is practically
always initiated by local breaks of vascular collagen. When there is vascular collagen
breakdown, platelet will encounter and readily to aggregate. Platelet aggregation will be
followed by activation of coagulation cascade leading to subsequent thrombus formation.3 When
thrombosis is occlusive and happened on atherosclerotic plaque near by coronary artery it could
stop up the blood supply into the heart leading to ischemia and heart attack.4 Hence, pathologic
vascular collagen degradation is a critical event determining the clinical symptom of ACS.
There was speculated that the benefit effect of coffee was due to antioxidant and antiinflammatory compounds, such as polyphenol, that is rich in coffee [Moreira]. The effect of
polyphenol-rich food or polyphenol extracts on development of atherosclerotic lesion had been
studied in animal model [mura, Vinson, dufi, manach, bernatova]. Polyphenol improved function
of endothelial cell and inhibit platelet aggregation (Vita 2005), decreased oxidation of LDL [de
whalley, Natella], improved cardiovascular remodeling and vascular function [bertanova],
increased plasma antioxidant carotenoid [svilaas]. In the present study, we speculated that
antioxidant and anti-inflammatory compounds in coffee might modulate inflammatory response
and enzymes that responsible for collagen degradation, it is matrix metalloproteinases (MMPs).
Matrix metalloproteinases is produced as inactive enzymes (zymogen) proMMPs, their
activation depends on any activators such as proteinases and or reactive oxygen species (ROS).
Inflammatory response increased production of inflammatory cytokines such as TNF-α, IL-1β
that can induce expression of proMMPs. In addition, bacterial infection induced large amount of
ROS that could activate proMMP by mechanism of cysteine switch. Therefore interaction
components in inflammation could result in MMP activation leading to degrade collagen.
The most significant result of this study was the influence of coffee consumption on the
morphology of collagen intimal. It may open a novel perspective study. There is only a few study
or literatures that mention the role of coffee in collagen metabolism, particularly vascular
collagen. Further studies are needed to elucidate this mechanism effect of coffee in collagen
metabolism, in addition, the influence of its structure to cardiovascular function as well. Like as
mention above, in addition to caffeine, coffee is a mixture of thousand substances including
antioxidants. There is still unclear, whether the effect of coffee on collagen metabolism was
mediated by its antioxidant, many works however are still needed to elucidate the role of
antioxidants in collagen metabolism.
The clinical significant of coronary artery morphology that composed by intact and dense intimal
collagen might provide protective effect against atherosclerosis and rupture. Since
atherosclerotic plaque rupture mostly occurred in area of thin collagen fibrous cap [Gouge,
2006]. Atherosclerotic plaque rupture is the proximate event of clinical manifestation of acute
coronary syndrome. Therefore it can be stated that coffee might prevent the incidence of acute
coronary syndrome by improving the morphology of arterial wall particularly intimal collagen.
Cardiovascular effect of coffee is necessary to continue to be explored. Although some studies
revealed the positive cardiovascular effect, many studies however suggested the adverse effect.
There is still common perception that caffeine in coffee can cause blood pressure to increase. This
is due to the effect of coffee to increase adrenal glands to release more adrenaline, unfavorable effect on
endothelial function (Papamitchel 2006, buschemy 2010) In addition to chemical compositions, variety
factors could influence the effect of coffee, such as frequency of consumption, type of coffee
procecesing, brewing method, host factors (e. g. age, gender), etc.
The present study use pure one dose of coffee decocta per day that represent one cup of coffee.
Further study are needed to analyze effect of the dosis, since some study indicated that high
intake of coffee have negative effect on cardiovascular health. In addition to dosis, coffee
serving usually was mixed with other ingredients such as milk and sugar. There still not known
whether coffee-nutrient interaction would influence the effect of coffee. Other factors like high
lipid diet, alcohol consumption, smoking are also commonly follow the habitual coffee
consumption are needed to study as well.
In conclusion, coffee consumption improved morphology of arterial wall leading to protection
against coronary atherosclerosis. This protective effect might also explain the resistance
mechanism of vasculature against rupture, thrombosis, and it could be meant that coffee
consumption could reduce the incidence of acute coronary syndrome. Most interestingly, this
study provide evidence that coffee consumption influence the structure of intimal collagen of
coronary artery. Further studies will be focused on elucidation of factors that affect its collagen
structure and composition.
Acknowledgement
This study was funding by University developmental grant of Jember University. Special thank
to Mbak Wahyu, Mas Agus and Mas Pinardi for helping us in laboratory works.
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